EXPERIMENT 1(A)

AIM : Enumeration of Red Blood Cells(RBCs) using haemocytometer.

MATERIALS REQUIRED : Neubauer chamber, RBC pipette, Cover slip, Needle, spirit,
cotton,
RBC diluting fluid
 Mercuric chloride : 0.5gm
 Sodium chloride : 1.0gm
 Sodium sulphate : 5gm
 Distilled water : 200cc
INTRODUCTION : The red blood cells or erythrocytes are circular, biconcave, non nucleated
cells containing haemoglobin and are embedded in blood plasma. After birth bone marrow is
the main site of formation of red blood corpuscles. These are involved in acting as a carrier of
oxygen and carbon dioxide. RBCs also maintain the ionic balance of human physiological
system and maintains viscosity of blood. Various pigments like bilirubin and biliverdin are
derived from RBC after their degradation.
PRINCIPLE : The basic principle is that the blood specimen is diluted (usually 200 times)
with red cell diluting fluid which does not remove the white blood cells but allows the red cells
to be counted under magnification in a known volume of fluid. Finally, the number of cells in
undiluted blood is calculated and reported as the number of red cells/µl of whole blood.
PROCEDURE : 1. Sterilize the finger tip with cotton plug soaked in spirit and let it dry.
2. Take a bold prick with needle to have free flow of blood and draw the blood in a RBC pipette
up to 0.5 mark.
3. Dip the RBC pipette in red blood cell diluting fluid and suck up diluting fluid up to 101 mark.
4. Rotate the pipette equally in your hands to mix the solution well by swirling.
5. Take the haemocytometer and place it on the flat surface of the work bench. Place the cover
slip on the counting chamber.
6. Allow a small drop of diluted blood, hanging from the pipette, to sweep into the counting
chamber by capillary action. Make sure that there is no air bubble and the counting chamber
must not be flooded.
7. Leave the counting chamber on the bench for 3 minutes to allow the cells to settle. Observe
the cells by placing the counting chamber on the mechanical stage of the microscope.
8. Focus on the centre room of the chamber and start counting the cells from upper left corner of
the room. It is advisable to complete all counts of the four squares and then move to the centre
square, which is the fifth square to be counted.
OBSERVATION :
SQUARE RBC
Square 1
Square 2
Square 3
Square 4
Square 5
Total

CALCULATION :

RBCs/cmm = No. of cells X Dilution factor X Depth factor X Total ruled area
Area count
Where; Dilution factor = 200; Depth factor = 10; Total ruled area = 25;
Area count = 5

RESULT: The number of red blood cells present in one µl of blood sample is___________
CLINICAL SIGNIFICANCE : The red cell count is the number of red cells present in one
cubic millimeter of blood. The normal values of the red blood cell count are:
 Woman : 4-5.5 million per cubic millimeter
 Men : 4.5-6.0 million per cubic millimeter
 Infants : 5- 6.5 million per cubic millimeter
Variations in normal values is observed in pregnancy, severe burns, diseased conditions and it
also depends upon altitude. It drops below normal values in anaemia and leukemia and rises
above the normal values in polycythemia and dehydration conditions. Therefore, the red cell
count is useful in diagnosis.
PRECAUTIONS : 1. First drop of blood should be discarded.
2. Pipette should be rinsed with anti coagulant before and after the experiment.
3. Blood and heme’s fluid should be taken up to the mark.
4. Mixing of blood and fluid should be done properly.
5. Complete circular structures should be counted.
6. Margin rule should be followed.
EXPERIMENT 1(B)
AIM : Enumeration of WBCs using haemocytometer .
MATERIALS REQUIRED : 1. Haemocytometer with New improved Neubauer
chamber,WBC pipette,Lancet,Cotton swab dipped in alcohol
WBC Diluting fluid
o Glacial acetic acid : 2.0 ml
o 1% Gentian violet : 1.0 ml
o Distilled water : 97.0 ml
This solution is stable at room temperature (25°C ± 5°C). A pinch of Thymol may be added as a
preservative.
Compound microscope.
THEORY : The white blood cells (WBCS) or leukocytes are nucleated, colorless blood cells.
They are originated purely from extra-vascular tissue. They are composed of nucleoproteins and
varieties of enzymes. Their number is less and life span is short as compared to red blood cells.
The WBCs exist in two forms viz. granulocytes and agranulocytes. Granulocytes are further
classified as eosinophil, basophil, neutrophil, while agranulocytes shows lymphocytes and
monocytes. These varieties possess independent morphological, functional and staining
properties. The main function of white blood corpuscles is phagocytosis that is body defence
mechanism against foreign particles and invading bacteria. They are also involved in antibody
formation in immunological body defence mechanism. It also take part in process of repair in an
area of inflammation.
PRINCIPLE : The blood specimen is diluted to 20 times of its original conc. with the WBC
diluting fluid and the cells are counted under low power objective of the microscope by using a
counting chamber. Blood is diluted with a solution called Turk’s fluid that causes the lysis of
RBCs and accentuates the nuclei of the white cells. This makes the counting of the white cells
easy. Turk’s fluid is a solution of glacial acetic acid and a dye gentian violet in distilled water.
The glacial acidic acid lyses the red cells while the gentian violet slightly stains the nuclei of the
leukocytes. The number of cells in undiluted blood is reported as per cu.mm (µl) of whole blood.
Blood cell counts can be performed using the hemacytometer.
[A] Haemocytometer: The hemocytometer is a thick glass slide, with its own cover-slip, which
larger than a regular cover slip. There are marked grooves that appear like an ‘H’. The
horizontal line of the ‘H’ separates the 2 grids for counting. Therefore, each slide has two
identical grids for counting cells. The depth of these 2 grids is a fixed 0.1mm
Counting Grids- Neubauer’s Chamber:Each grid is a square with the dimensions of 3×3 mm2.
This square has three equidistant vertical and horizontal lines. These divide it into 9 smaller
squares of 1×1 mm2 each. These are separated from each other by triple-ruled lines. Of these 9
squares, the 4 corner squares are used to count bigger cells, like WBCs, while the center square
is used to count smaller cells, such as RBCs.The 4 corner squares of the main grid are further
divided into 16 smaller cells. The center square of the main grid is divided into 25 smaller
squares, each of which is again divided into 16 smaller squares. The sample to be counted is
loaded onto the slide after the coverslip has been placed. Excess fluid drains into the grooves on
the side. However, the person loading the sample must be extremely careful while loading.
WBC Counting rules: When WBCs are counted, the calculation is much easier. WBCs are
counted in the 4 corner squares of the main grid. These squares have an area of 1 mm2 each. To
get the WBC count, the number of cells in each square are counted, and their mean is then
calculated. Let the mean be ‘n’. The volume of each square is 1 x 0.1 = 0.1 mm3. The number
of cells in 1 mm3 is n/0.1.
Therefore, the total number of cells in 1ml is (n/0.1) x 1000. We multiply by one thousand as
1000 mm3 = 1 cm3; and 1 cm3 = 1 ml
[B] WBC pipette: This pipette (also called Thoma pipette) long stem is divided into two parts:
A glass tube and a flexible rubber tube.
 The long stem of the glass tube is marked with 0.5 and 1.0
 While the short arm after the bulb is marked 11.
 Its central portion is a bulb or a globular shape with one white bead in it.
 Rubber tubing is attached to suck the blood.
The dilution of the blood to the TLC fluid is 1:20.
PROCEDURE : Preparation of WBC diluting fluid (Turk’s fluid):
Turk’s fluid a 2% solution of acetic acid. Add 2 ml of glacial acid, 1 mL gentian violet (1 %),
and add 97 ml of distilled water to make up to 100 ml solution.
Total leucocytes counting Procedure:
1. Sterilize the fingertip with cotton plug soaked in 70% alcohol and let it dry.
2. Take a bold prick to have free flow of blood.
3. Draw the blood in a WBC pipette up to 0.5 mark.
4. Dip the WBC pipette in WBC diluting fluid up to 11 mark.
5. Take the haemocytometer and place it on the flat surface of the work bench.
6. Place the cover slip on the counting chamber.
7. Allow a small drop of diluted blood, hanging from the pipette, to sweep into the counting
chamber by capillary action.
8. Make sure that there is no air bubble and there is no overfilling beyond the ruled area.
 9. Leave the counting chamber on the bench for 3 minutes to allow the cells to settle.
10.Observe the cells by placing the counting chamber on the mechanical stage of the
microscope.
11.Focus on one of the corner squares of the counting chamber and count the white cells
schematically, starting from the upper left small square of each Square.
12.Repeat the count in all the four corners of the chamber.
13.Apply the margin rules i.e. count the cells lying on left and top margins, and discard
those on the right and bottom margins.
CALCULATIONS : 1. Count the cells in the Neubauer chamber. These are counted in the four
large corner squares labeled as WBC and if the number is Y.
2. Calculate on the basis of following dimensions:
3. One large area is 1 x 1 mm, and the depth is 0.1 mm.
4. Total area counted in 4 large squares = 4 x 1 x o.1 = 0.4 µL (4/10).
5. Y x 10/4 is the total WBC in the cell in 1 µL.
6. Now dilution is 1:20.
7. Number of WBC in 1µL = Y x 10 x 20/4 = Y x 50 = Total WBC count.
8. Total TLC = counted cells (Y) x 50 = TLC/cmm.

RESULT : The Total Leucocyte Count (TLC) in the blood sample is ___________ / mm3 of
blood.
SOURCE OF ERRORS :
 If there are micro-clots in the sample.
 If inadequate mixing is done.
 Improper filling of the chamber.
 Presence of air bubbles in the pipette.
 If the dilutions are improper.
 Mistakes in the calculations.
CLINICAL SIGNIFICANCE: The white cell count is the number of white cell present in one
cubic millimeter of blood. The normal values of white blood cell count varies between 5000 to
10,000 per cubic millimeter or 7-11 thousand cells/µl of blood volume in healthy individual.
Variations in normal values is observed in diseased states. WBC count increases (leucocytosis)
in conditions like pneumonia, leukemia, meningitis, small pox etc. while the count decreases
(leucopenia) in conditions such as influenza, typhoid, infectious hepatitis etc. Moreover the
count rises in pregnancy and during menstruation. Thus, white blood cell count is useful in
diagnosis.
Normal values of TLC:
 Adult /child = 5000 to 10,000 /cu mm of blood or /µl of blood
 Child ≤2 years = 6200 to 17000 /cu mm. of blood
 Newborn = 9000 to 30,000 / cu mm. of blood
Change in TLC levels
 At birth = 10,000 to 25,000/ cu mm. of blood
 Infants = 8000 to 15,000/ cu mm. of blood
 Adults = 4000 to 10,000/ cu mm. of blood
 Pregnant ladies = 12,000 to 15,000/ cu mm. of blood
Critical value = <2500 or >30,000 /cmm ( These are panic value).
PRECAUTIONS : 1. First of blood should be discarded.
2. Pipette should be rinsed with anti coagulant before and after the experiment.
3. Blood and turk's fluid should be taken upto the mark.
4. Mixing of blood and fluid should be done properly.
5. Complete circular structures should be counted.
6. Margin rule should be followed.